Figure 2.
Identification of lead CH3 mutations for Fc heterodimer formation using non-reducing SDS-PAGE analysis. Non-reducing SDS-PAGE of representative Fc engineered antibodies are shown in (A). Protein samples were harvested. Gel was stained with Coomassie blue. Mutations that induce the formation of half-antibody fragments are circled. These include the published knobs-into-holes mutations (lanes 4 & 5) and the computationally engineered variant (lane 6).(B) Identification of compensatory Fc mutations using a three-chain system (2 heavy and 1 light). The Fc mutations shown in column 7 (circled in red) show maximum reduction in the formation of half-antibody fragments, similar to the traditional knob-into-holes mutations (column 3). Lane 1 in A and B contains SeeBlue protein standards.